The laboratory is interested in understanding the clathrin-independent endocytosis pathway that is associated with Arf6. We have shown that this pathway is responsible for internalizing plasma membrane (PM) proteins that lack sequences that allow recognition by the clathrin and adaptor protein machinery. Among the proteins that enter cells via this mode of endocytosis are the major histocompatibility complex Class I protein (MHCI) and the lipid anchored protein CD59. After endocytosis, MHCI and CD59 in vesicles are delivered to and fuse with endosomes containing cargo proteins from the clathrin-dependent endocytosis pathway such as transferrin receptor. From there, MHCI and CD59 can proceed on to late endosomal compartments where they are degraded or they can be recycled back out to the PM via unique tubular recycling endosomes. In HeLa cells these recycling endosomes contain only cargo that had entered via clathrin-independent endocytosis and their return to the PM is dependent upon the activity of Arf6 and several other regulators including Rab11 and 22. We have identified 7 new cargo proteins that enter cells and traffic along this clathrin-independent pathway: CD44, CD55, CD98, CD147, Lat1, ICAM1 and the non-insulin stimulated glucose transporter Glut1. We have confirmed that these proteins enter cells by clathrin-independent endocytosis in many human cell lines and in addition, in HeLa cells, we see a difference in intracellular itinerary followed by these proteins. CD55 and Glut1 follow a similar itinerary as MHCI, merging with endosomes containing the transferrin receptor before routing to the tubular recycling endosomes. By contrast, CD44, 98 and 147 after endocytosis do not enter transferrin-containing endosomes but are routed directly to the recycling endosome. This divergence in itinerary suggests that clathrin-independent cargo proteins are sorted within the cell and we are studying whether signals contained on these cargo proteins (extracellular, transmembrane or cytoplasmic) are directing their trafficking. We have found that sequences in the cytoplasmic tail of CD147 are responsible for this altered itinerary and through a yeast two-hybrid screen are looking for proteins that recognize these cytoplasmic sorting sequences. We are also examining the action of the MARCH E-3 ubiquitin ligases, which when overexpressed in cells leads to the downregulation of a number of clathrin-independent cargo proteins including MHCI. The laboratory has also been developing new probes for following endocytosis using SNAP-tag technology and has filed a provisional patent application for making releasable SNAP-tag ligands. This technique could be useful for quantitative evaluation of endocytosis and recycling of specifically labeled cell surface proteins. In addition to regulating recycling of endosomal membrane back to the cell surface, Arf6 has also been implicated in some forms of regulated secretion. Both of these processes generally require the actin cytoskeleton for exocytosis of the membrane back to the plasma membrane. In a collaborative study, it was found that myosin Vc is associated with tubular endosomes in breast cancer cell line MCF7 and also with secretory granules (Jacobs et al 2009). Although it is not clear whether myosin Vc is associated with the recycling endosomes in HeLa cells, we are investigating roles for other motor proteins, including microtubular-based motors, in this process. Finally, we have found that clathrin-independent endocytosis is observed in a variety of cell types and found evidence that this form of endocytosis participates in lumen formation observed in human vascular endothelial cells cultured in collagen gels, a model for vasculogenesis.